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Fig. S1

In vitro assay for the cleavage by AtSBT5.4-myc of a fluorogenic peptide representing the putative cleavage site for CLV3. AtSBT5.4-myc was pulled down from transgenic plant extracts with anti-myc beads. 40 µl of bead-bound AtSBT5.4-myc were added to a solution containing a final concentration of 50 µM fluorogenic peptide in a buffer consisting of 25 mM MES-sodium acetate (pH 5.5) supplemented with 2.5 mM CaCl2. Reaction kinetics were monitored by fluorescence at 420 nm (10 nm slit) from excitation at 320nm (10nm slit) in a BioTek Spectrophotometer. Control peptide contains canonical subtilase core recognition sequence (RRXL). CLV3 peptide contains sequence at putative cleavage site. Empty vector control represents pull down of an extract from transgenic plants transformed with an empty vector. Cont pep + enz = control peptide: Abz-RRILEGLPY(NO2)D-OH + AtSBT5.4-myc; CLV3 pep + enz = CLV3 peptide: Abz-LHEELRTVPSY(NO2)D-OH+AtSBT5.4-myc; Cont pep = control peptide alone; Cont pep + emp vector = control peptide + empty vector extract.

Fig. S2

In vitro assay to test for the cleavage of CLV3 by AtSBT5.4. AtSBT5.4-mycwas affinity purified from transgenic Arabidopsis plants. 500 g of seedlings were ground in liquid nitrogen and suspended in 25 mM Tris-HCl, (pH 7.2), 150 mM NaCl, 0.1% Nonidet P-40 and 10% glycerol. 250 l of anti-c-myc agarose conjugate (A7470 from Sigma) were added to the filtered lysate and incubated for 2 h at 40C with continuous rotation. The agarose beads with bound AtSBT5.4-mycwere recovered by centrifugation at 1000 g for 3 min at 40C. The beads were washed twice with washing buffer (25 mM Tris-HCl (pH 6.0), 150 mM NaCl) and then with 25 mM Tris-HCl, 25 mM MES, 25 mM acetic acid (pH 6.0). Parallel purification was performed using transgenic plants transformed with the empty vector to obtain material for control reactions.

The his-tagged fragment of CLV3 was used as substrate for AtSBT5.4-myc cleavage reactions.Two primers CLVF (CGGGATCCATGTCCGGTCCAGTTCAACA) and CLVR(TTCCTTTTGCGGCCGCAAGGGAGCTGAAAGTTGTTTCTT ) were used to amplify the CLV3, and the PCR products were cloned into pET28a at BamHI and NotI sites. The his-CLV3 fragment was expressed in and purified from E. coli (BL-21 cells) using Ni-NTA Agarose (Qiagen) column chromatography according to the manufacturer’s instructions. Reactions (50l) contained 100 ng of his-CLV3 fragment and 35 µl of bead-bound AtSBT5.4-mycin an assay buffer comprised of 25 mM Tris-HCl (pH 6.0), 25 mM MES (pH 6.0), 25 mM acetic acid, 1 mM CaCl2. The reaction was carried out at 320 C and stopped by addition of 2 mM EGTA. Cleavage activity was assayed by SDS-PAGE and Western blot analysis using anti-HIS antibody (Amersham Biosciences).

Fig. S1

Fig. S2